Herpes Simplex virus, type 1 (HSV-1) causes human specific infections in the form of recurrent cold sores and corneal keratitis, and even blindness with periods of latency in neurons that serve as reservoirs of lifelong infection. The infection is age dependent, with exposure rates exceeding 70% in older US adults. HSV-1 encephalitis is rare, but it can be fatal. Though currently available treatments (e.g., acyclovir / valacylovir) are efficacious for acute HSV1-induced lytic infections, they do not eliminate latent infections. Most of our knowledge regarding HSV-1 latency comes from small animal models, which are not naturally infected by HSV-1. Furthermore, these animal models do not fully recapitulate the species-specific effects of human HSV- 1 infection. Published human models have relied largely on neurons derived from human neoplastic cells, but they may not fully reflect the global mRNA expression profile of normal human neurons. Recently, human fetal dorsal root ganglion cells have been employed, but the difficulty in obtaining aborted fetuses limit their use. Human induced pluripotent stem cells (iPSCs) technologies offer an unprecedented opportunity to generate unlimited supplies of human neurons and the facility to manipulate such cells in vitro. We have developed an in vitro HSV-1 infection model in human iPSC-derived neurons (hiPSCs-neurons) that displays acute lytic infection as well as quiescent infection with features similar to the animal models. Our method to differentiate human neurons from iPSCs is robust, and it enables in a cost effective way, an adequate number of cells required for chromatin analysis. The central tenet of this proposal is that an understanding of the regulatory mechanisms HSV-1 infection in human cells will highlight the pathways for the development of drugs with highest therapeutic efficacy. We will employ our iPSC-based in vitro human model of HSV-1 infection to investigate aspects of the heterochromatinization of viral episomes during quiescence and to characterize HSV-1 miRNAs. In the Aim 1 of this proposal, we will perform chromatin analysis of HSV-1 genomes in acutely and latently infected hiPSCs-neurons. In the Aim 2, we will characterize HSV-1 miRNAs in HSV-1 infected human neurons, investigate their functions, and identify their human RNA targets. The anticipated outcomes from this proposal are: (i) improved understanding of human neuronal responses to lytic and quiescent HSV-1 infection; (ii) identification of targets that will ultimately pave the way for the identification effective anti-viral drugs with a novel mechanism of action.